基于DNA-SIP技术的污水氨氧化微生物系统发育与生理代谢功能关系的研究

Microbial ammonia oxidation is the first and rate-limiting step of nitrification. For more than 100 years it was believed that Ammonia-Oxidizing Bacteria (AOB) were the only group responsible for the oxidation of ammonia. However, recent findings expanded this pathway also to Ammonia-Oxidizing Archaea (AOA). In our studies (NSFC. 51078007), the abundance and composition of AOA and AOB of 50 full-scale wastewater treatment plants (WWTPs) in China have been investigated using quantitative real-time polymerase chain reaction (PCR), cloning and sequencing approaches based on monooxygenase subunit A (amoA) genes. However, the methods and results only give us the phylogeny information of AOA and AOB in wastewater treatment, they do not necessarily enable us to infer their functions. One of the biggest challenges that microbial ecologists face is "Who eats what, where and when?". New technologies like DNA/RNA based Stable Isotope Probing (DNA/RNA-SIP), is increasingly being used in attempts to link the identity of microorganisms to their functions. So, in this project, the linking between phylogenies of AOA, AOB and their metabolic functions in wastewater treatment will be investigated using DNA/RNA-SIP technique. On the basis of previous studies (NSFC. 51078007), typical WWTPs will be chosen. Then through in field label or microcosm incubation, 13C-labeled substrate DNA/RNA-SIP will be employed to reveal whether AOA in wastewater treatment is autotrophic or heterotrophic, whether nitrification was driven by AOA or AOB, the relative contributions of AOA and AOB to ammonia oxidation, AOA's metabolic pathway, activity of AOA and AOB during nitrificaiton, and the temporal and spatial heterogeneity of AOA and AOB. And time-course DNA-SIP will be used to systematically investigate the effects of environmental factors such as ammonium levels, inhibitors, pH value, alkalinity, organic carbon, temperature, salinity, DO levels and sludge retention time on the growth of AOA and AOB, changes in AOA and AOB abundance and composition, activities of AOA and AOB, the linking between phylogenies of AOA, AOB and their metabolic functions. A combined approach of 13C-labeled substrate DNA/RNA-SIP, catalyzed reporter deposition-FISH (CARD-FISH) and stable isotope tracer technique of 15N and 18O will be employed to detect the potential metabolic coupling of AOA and AOB in nitrification/denitrification, short-cut nitrification/denitrification, simultaneous nitrification and denitrification and anaerobic ammonium oxidization process. On the basis of this project, the linking between phylogenies of AOA, AOB and their metabolic functions in wastewater treatment will be better understood. Furthermore this project will provide microbial foundation for regulating and developing novel biological nitrogen removal process.

氨氧化是生物硝化的限速步骤。申请者对"50个不同污水生物脱氮工艺中AOA和AOB的种群数量和组成特征"（51078007）进行了系统研究，但是发现现代分子生物学技术无法解决AOA和AOB系统发育与生理代谢功能之间的耦合这一关键问题。因此拟利用稳定同位素核酸探针技术（DNA/RNA-SIP）研究原位状态下AOA、AOB系统发育与生理代谢功能之间的关系。对典型实际污水厂，以DNA-SIP技术为核心，结合RNA-SIP、稳定同位素示踪和FISH技术，探究原位条件下AOA和AOB的氨氮和碳的代谢途径、营养类型、活性、时空异质性、系统发育与贡献的关系，考察环境因素和工艺条件对两者系统发育和代谢功能关系的影响，研究两者在全程脱氮、短程脱氮、同时硝化反硝化及Anammox工艺中的相互作用机制。为充分利用AOA和AOB，开发新型高效的污水脱氮工艺提供微生物学基础和技术指导。

利用DNA-SIP技术解决AOA和AOB系统发育与功能之间关系这一关键问题,发现:Na213CO3为DNA-SIP培养的唯一碳源,直接证明活性AOA和AOB均为自养生长;AOA和AOB在不同条件下单独或同时执行氨氧化,活性AOA主要为Nitrososphaera:含Ca. Nsp. gargensis、Nsp. sp. JG1、Nsp. viennensis、Nsp. evergladensis等,Nitrosopumilus为次要活性AOA;活性AOB主要是Nitrosomonas;丰度大,不代表其一定为活性AOMs;活性氨氧化微生物(AOMs)存在时间和温度异质性;低氧、高盐度、高浓度Al2O3\TiO2长期影响等恶劣环境下AOA是主要活性AOMs。Comammox普遍存在于WWTPs中。针对WWTPs常见AOA设计qPCR引物;将GelGreen成功运用于DNA-SIP重层DNA鉴定。.AOMs生境微生态的研究中:某WWTP一年细菌丰度和多样性动态变化不明显,持久性细菌多样性较低但相对丰度很高;16个WWTPs细菌多样性差异较大,处理混合污水的细菌多样性较生活污水和工业废水高;AOA和Comammox是某城市PM2.5氨氧化的重要贡献者;某地地下25至1900m土壤中,DNA-SIP发现AOA和AOB均参与氨氧化。.WWTPs原位DNA-SIP发现:AOA丰度占优势的某WWTP中,AOA是活性AOMs;某WWTP三期工艺一年研究发现,AOA丰度略大于AOB,但AOB是主要活性AOMs;在23个WWTPs中,AOA和AOB丰度差异不大,AOA是冬季主要活性AOMs;在13°C和25°C时,AOA和AOB是活性AOMs;而AOA是37°C时的活性AOMs;某WWTP中,AOA和AOB是11、12月份主要活性AOMs,但在1月份是AOA;低氧条件下CANON系统中AOA是活性AOMs。.环境因素和工艺条件DNA-SIP发现:不同盐度下AOA始终是活性AOMs;某厂环境浓度TiO2时AOB是活性AOMs,在高浓度TiO2下AOA是活性AOMs;AOA是某厂种泥及不同浓度纳米TiO2和Al2O3长期作用下的活性AOMs;7000 µg/L的ROX长期作用导致亚硝积累,ROX长期作用下,AOA和AOB均参与氨氧化过程。.本研究为开发新型高效的污水脱氮工艺提供微生物学基础。